Anti-mucus glycoprotein monoclonal antibody

ABSTRACT

An IgM class monoclonal antibody which specifically reacts with mucus glycoproteins produced by human gastric gland-type mucous cells is provided. By performing immunohistochemical staining using a labeled derivative of the monoclonal antibody, human gastric gland-type mucous cells as well as mucus secreted by these cells can be specifically stained. The monoclonal antibody can also be used for the analysis of gastric gland-type mucous cell-derived mucus glycoproteins in human body fluids as well as for the examination or diagnosis of cancer.

FIELD OF THE INVENTION

The present invention relates to a novel monoclonal antibody which canbe used in assays for human gastric gland-type mucus. Thus, the presentinvention provides a novel antibody which has strong affinity for humangastric gland mucous cells as well as for the mucus secreted thereby butwhich has cross-reactivity neither with human gastric surface mucouscells nor with the mucus secreted thereby.

BACKGROUND ART

The mucus-secreting cells present in the epithelium of alimentary tractmucosae including gastric mucosa are considered to be involved in themaintenance of gastrointestinal functions by synthesis, followed bysecretion, of mucus. Gastric mucus in particular is viewed as one of theimportant defense factors to protect the gastric mucosa from attack bysuch factors as gastric acid and pepsin.

The mucus production in the human stomach is effected by surface mucouscells (covering epithelial cells) present in the gastric mucosa surfaceepithelium and gastric gland mucous cells present in the deep part ofthe gastric gland. Recent years have seen attempts to differentiateindividual mucus species produced by these mucous cells. A histochemicalstaining method has been developed which is specific for the sugarcomponents peculiar to the respective mucus species of surface mucouscells and gland mucous cells, and hence is able to differentiate betweenthem. Thus, the mucus found in the surface mucous cells can bespecifically stained by the galactose oxidase-cold thionin Schiff(GOCTS) reaction, while in the gland mucous cells mucus classified asclass III by the Concanavalin A paradoxical staining method (Katsuyama,T. and Spicer, S. S. (1978), J. Histochem. Cytochem. 26, 233-250) isfound to be specifically localized (Ota, H. et al. (1991), HistochemicalJ. 23, 22-28). Mucus gel layer covering the surface epithelium of thegastric mucosa has been proved to be a laminar structure of alternatinglayers of the GOCTS-reactive mucus and the Class III mucus, and themucus gel layer has been found to contain both mucus species fromsurface mucous cells and gland mucous cells (Ota, H. et al. (1991),Histochemical J. 23, 22-28).

Katsuyama et al. have elucidated that in normal tissues class III mucuscan be found only in the limited glandular epithelia of the alimentarytract ranging from the cardiac glands of the esophagus through thecardiac glands, mucous neck cells and pyloric glands of the stomach,Brunner's glands of the duodenum, the mucous glands of the papilla ofVater and the mucous glands of the pancreatic duct in the head of thepancreas, whereas it can be demonstrated with high incidence in suchtumor tissues as in cholecystic adenoma, gallbladder cancer andpancreatic duct cancer (Katsuyama et al. (1989), Byori to Rinsho 7,1217-1224). Furthermore, Matsuzawa et al. has found, referring to therelationship between metaplasia and cancer, that such forms of tissue asfound in the gastric mucosa where strongly GOCTS-positive mucus is foundin the surface epithelium of the mucosa and class III mucus is localizedin the deep part thereof cannot be seen in normal pancreatic ducttissues but frequently in pancreatic duct tissues with metaplasia orcarcinoma (Matsuzawa, K. et al. (1992), Human Pathology 23, 925-933).These study results suggest that data for the diagnosis of cancerdiseases or possible cancerization can be presented by determining thegastric type mucus secreted into the body fluid, particularly thegastric gland-type mucus.

As for the specific detection of the human gastric gland-type mucus, noother methods than the above-mentioned Concanavalin A paradoxicalstaining method have been known to date. The known detection method,however, cannot quantitatively determine the gastric gland-type mucusbecause of its poor reproducibility of staining and also because of thedrawback that the method can be applied only to fixed specimens andhence cannot be applied to the determination of secreted or solubilizedmucus.

DISCLOSURE OF THE INVENTION

As a result of their intensive studies of monoclonal antibodies capableof recognizing the human gastric gland-type mucus, the present inventorshave now succeeded in obtaining a hybridoma producing a novel monoclonalantibody with high specificity for human gastric gland-type mucous cellsand for the mucus secreted therefrom, as well as the monoclonalantibody, by using as antigen for immunization mucus glycoproteins(mucins) which are the major components of the mucus.

Thus, the present invention provides, as described in 1.-5. below, anovel monoclonal antibody and hybridoma, as well as use of themonoclonal antibody for the analysis of gastric gland-type mucouscell-derived mucus glycoproteins contained in body fluids.

1. An IgM class monoclonal antibody which specifically reacts with mucusglycoproteins produced by human gastric gland-type mucous cells.

2. The monoclonal antibody of 1. wherein the IgM class monoclonalantibody is produced by a hybridoma deposited in the National Instituteof Bioscience and Human-Technology (NIBH), Agency of Industrial Scienceand Technology 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305,Japan, on Apr. 28, 1993 with the accession number P-13622.

3. A hybridoma which produces an IgM class monoclonal antibody withspecific reactivity with mucus glycoproteins produced by human gastricgland-type mucous cells.

4. A hybridoma deposited in the National Institute of Bioscience andHuman Technology (NIBH), Agency of Industrial Science and Technologywith the accession number P-13622.

5. Use of the monoclonal antibody of 1. for the analysis of gastricgland-type mucous cell-derived mucus glycoproteins contained in humanbody fluids.

The present invention thus relates to a monoclonal antibody useful forthe staining of gland mucous cells (i.e. cardiac gland cells, mucousneck cells and pyloric gland cells) of the human gastric mucosa as wellas mucous cells producing human gastric gland-type mucus (e.g. Brunner'sglands of the duodenum, gastric gland-type metaplastic cells and gastricgland-type tumor cells). Furthermore, the present invention relates to amonoclonal antibody useful for the determination of mucus glycoproteinssecreted by the above-mentioned gastric gland-type mucous cells. Moreparticularly, immunohistochemical staining with the monoclonal antibodyof the present invention or a labeled derivative thereof permitsspecific staining of human gastric gland-type mucous cells as well asthe mucus secreted thereby, and ELISA (enzyme-linked immunosorbentassay) therewith permits the analysis, both qualitative andquantitative, of gastric gland-type mucous cell-derived mucusglycoproteins contained in human body fluids, e.g. gastric juice,pancreatic juice, blood and sputum. The monoclonal antibody of thepresent invention can also be used for the examination and diagnosis ofcancer.

The hybridoma which produces the monoclonal antibody of the presentinvention may be obtained by any conventional method, e.g. the method ofKoehler and Milstein (Koehler, G. and Milstein, C. (1975) Nature 256,495-497), using mucus glycoprotein prepared by any appropriate method.Thus, the above-mentioned mucus glycoprotein is used to immunize a mouseand splenic cells from the mouse are fused with murine myeloma cells.From among the resultant hybridomas can be selected and isolated ahybridoma which produces and secretes the desired antibody. Methods ofpreparing the above-mentioned hybridoma and monoclonal antibody of thepresent invention, as well as characteristics of the monoclonalantibody, will now be described in the following.

A. Isolation and Purification of Antigen:

As antigen for immunization there may be used extracts from human ornon-human mammalian gastric mucosa or Brunner's glands. Preferably,mucus glycoproteins obtained from these extracts are purified prior totheir use. Thus, for example, the mucus glycoprotein extracted andpurified from rat gastric mucosa in accordance with the method of Oharaet al. (Ohara, S. et al. (1986), Comp. Biochem. Physiol. 83B, 273-275)is used. In the examples described below, the extracts from rat gastricmucosa were fractionated by gel filtration and fractions correspondingto molecular weights not lower than 1,500,000 are collected andsubjected to CsCl equilibrium density gradient centrifugation to affordglycoprotein fractions (mucus glycoprotein-containing fractions). Thethus obtained mucus glycoprotein was used as immunizing antigen.

B. Immunization of mice

As animals to be immunized, there may preferably be used four toeight-week-old BALB/c mice, although mice of other strains may also beused. Immunization schedule and antigen concentration are selected suchthat an adequate quantity of antigen-stimulated lymphocytes are formed.Thus, for example, mice are intraperitoneally administered with 100μg/animal of the above-described mucus glycoprotein together withappropriate adjuvant. Subsequently, the animals were administeredseveral times at intervals of several days or weeks with the sameantigen as used for the first immunization. After the secondimmunization, blood samples are taken from the eyeground vein to assaythem for antibody titre. Antibody titre may preferably be measured byELISA.

C. Cell Fusion

The spleen is excised from the immunized mouse and an aqueous suspensionof single splenic cells is prepared therefrom. The suspension issubjected to cell fusion with murine myeloma cells using an appropriatefusogenic agent. As a preferred example of such fusogenic agent theremay be mentioned polyethylene glycol of mean molecular weight 400-6,000,although any other fusogenic agents known in the art may also be used(Klebe, R. J. and Mancuso, M. G. (1981), Somat. Cell. Genet., 7,473-488). In the examples described below there was used polyethyleneglycol 4,000 (E. Merck; Cat. No. 9727). Those myeloma cells arepreferably used which are derived from the same animal species as theanimal used for the preparation of splenic cells and which are not toproduce any antibody. In the examples described below, there were used8-azaguanine-resistant mouse myeloma cells Sp2/O-Ag14 (Shulman, M. etal. (1978) Nature 276, 269-270). It is preferred to use splenic cellsand myeloma cells at a cell count ratio of about 20:1--about 5:1.

D. Selection of Fused Cells

In a separate container, the mixture obtained in C. above, i.e. themixture of unfused splenic cells, unfused myeloma cells and fused cellsis cultured for an appropriate period in selection medium which does notsupport the growth of the unfused cells, thereby to kill the unfusedcells. As such medium there may be used one which does not support thegrowth of unfused myeloma cells which are drug-resistant (e.g.8-azaguanine-resistant), for example, HAT medium. Since unfused spleniccells are non-tumor cells, unfused splenic cells and unfused myelomacells die in such selection medium after a certain period. Fused cellsare viable in such selection medium because they have both tumorcharacteristic of their parental myeloma cells and characteristics ofsplenic cells.

E. Identification of Antibody Produced by Hybridoma in Each Container(Screening)

The culture supernatant is sampled from each of the wells with respectto which hybridoma has been confirmed to be formed by the cultivation inD. above, and assayed for the presence of immunoreactivity with gastricmucus glycoprotein. Such screening may preferably be performed by ELISA,for example. Immunoreaction-positive samples are further screened forspecific recognition of gastric gland mucous cells and the mucussecreted thereby. Such screening may preferably be carried out, forexample, by immunohistochemical staining using sections of fixed gastricmucosa specimens.

F. Cloning of Hybridoma Producing the Desired Antibody and Production ofthe Antibody

The immunoreaction-positive cell suspension obtained in E. above is thencloned by any appropriate method, for example, by the limiting dilutionmethod, whereafter the desired antibody may be obtained in either one ofthe two ways. One way involves cultivation of hybridoma in anyappropriate medium for a certain period, followed by isolation from theculture supernatant of the monoclonal antibody produced by thehybridoma. The other way involves intraperitoneal inoculation of asyngeneic or semi-syngeneic mouse with hybridoma. In the latter case,the desired monoclonal antibody produced by the hybridoma may beisolated after a certain period from the blood or ascites fluid of theinoculated mouse.

G. Purification of Monoclonal Antibody

From the hybridoma culture supernatant, or from the mouse blood orascites fluid, as obtained in F. above, the monoclonal antibody of thepresent invention can be purified by any biochemical purification methodwidely used in the art. Thus, for example, such purification maypreferably be carried out by combining different purification proceduressuch as ammonium sulfate salting out, ion-exchange columnchromatography, molecular sieve column chromatography or affinity columnchromatography.

H. Labeling of Monoclonal Antibody and Use of the Labeled MonoclonalAntibody

The thus purified monoclonal antibody may be labeled by any biochemicalmeans widely used in the art with peroxidase, alkaline phosphatase,biotin etc. Labeling may be performed by linking product from periodateoxidation of the enzyme to the antibody or by combining the enzyme andantibody together using glutaraldehyde as crosslinking agent. The thusobtained labeled monoclonal antibody can be conveniently applied toimmunohistochemical staining or ELISA-based sandwich assay. As for theELISA method, the quantitative determination of gastric gland-typemucous cell-derived mucus glycoprotein contained in a sample may beperformed, for example, by adsorption of the monoclonal antibody of thepresent invention as primary antibody onto each well of an ELISA plate,followed by further adsorption of a material which is not involved inthe reaction, e.g. skim milk, onto the adsorbed sites of each well.Sample solution is then added to each well and the wells are washed.Aliquots of labeled monoclonal antibody solution of appropriateconcentrations are added to the wells, followed, after washing of thewells, by addition of a solution of substrate for the enzyme used in theELISA.

I. Reactivity of Monoclonal Antibody with Antigen

The antigen recognizing site of the monoclonal antibody according to thepresent invention can be examined by studying the reactivity of themonoclonal body with compounds of known structures. The presentinventors subjected crossreacting pig gastric mucus protein (Sigma) todecomposition treatment by conventional biochemical means and componentswith antigenic activity were separated and isolated from thedecomposition products and analyzed by conventional physicochemicalmeans. The antigenic activity may be conveniently measured by aCompetitive ELISA which is an application of the inhibition assay to theELISA.

The present invention will now be further illustrated by way ofexamples, to which the present invention is by no means limited.

Example 1

(1) Preparation of antigen for immunization

Stomachs were excised from female SD rats weighing 250-300 g (Japan SLC,Inc.) and the gastric mucosae were collected therefrom by scraping andthen subjected to extraction with 50 mM Tris-HCl buffer, pH 7.2,containing 2% of the non-ionic surface active agent TRITON X-100(polyethylene glycol mono p-isooctylphenyl ether). The extracts weresubjected to gel filtration using a Bio-Gel A, agarose gel beads, 1.5 mcolumn (Bio-Rad Laboratories) to collect the eluted fractions ofmolecular weights not lower than 1,500,000. These fractions were furthersubjected to CsCl equilibrium density gradient centrifugation (N.B.) toobtain purified glycoprotein antigen (mucus glycoprotein fraction) ofdensity 1.4±0.4 g/ml, using a fraction collector.

N.B. "CsCl equilibrium density gradient centrifugation" was carried outunder the following conditions:

    ______________________________________                                        Centrifugation     1.5 × 10.sup.5 g                                     Temperature        10° C.                                              Period             85 hours                                                   Apparatus          Model 72P;                                                                    Rotor : RPS-40T                                                               (Hitachi, Ltd.)                                            ______________________________________                                    

(2) Preparation of splenic cells from immunized mice

Four-week-old female BALB/c mice were immunized intraperitoneally with50 μl of Freund's complete adjuvant (Difco, Laboratories) and 100μg/animal of the purified mucus glycoprotein antigen obtained by theprocedure mentioned above (hereinafter referred to as the purified mucusglycoprotein antigen). The animals were then intraperitoneallyadministered, at 3-week intervals for boosting, with 50 μl of Freund'sincomplete adjuvant (Difco, Laboratories) and 100 μg/animal of the samepurified mucus glycoprotein antigen as used for the first immunization.On the third or fourth day after each boosting, blood samples were takenfrom the eyeground vein and analyzed for antimucus glycoprotein antibodyin the sera by the ELISA method as described below.

ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05Msodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at aconcentration of 2 μg/ml and the same buffer was used to preparetwo-fold serial dilutions. 100 μl aliquots of each dilution was added towells of a microplate adapted for use in ELISA (Corning) and the wellswere allowed to stand overnight at 4° C. Each well was washed threetimes with 0.05% of the non-ionic surface active agent TWEEN-20,polyoxyethylene sorbitan monolaurate, (Nacalai Tesque, Inc., Japan) inPBS (PBS-TWEEN), filled with 2% skim milk in PBS, and then allowed tostand for 1 hour. The wells were washed three times with PBS-TWEEN and a1,000-fold dilution of mouse serum sample was dispensed in 100 μlaliquots into the wells. After incubation for 1 hour, the wells werewashed three times with PBS-TWEEN, and 100 μl of a 10,000-fold dilutionin PBS of peroxidase-labeled goat anti-mouse immunoglobulins antibody(Tago, Inc.) was added as secondary antibody to each well and allowed tostand for 1 hour. The wells were washed three times with PBS-TWEEN and100 μl of an ABTS(2,2'-azino-di[3-ethylbenzthiazoline sulfonate (6)])-H₂O₂ peroxidase substrate solution (Kirkegaard & Perry Laboratories) wasadded to each well. After reaction at room temperature for 30 minutes,the optical density at 415 nm was measured for each well using amicroplate reader. Those mouse sera which developed a color independence on the dose of the mucus glycoprotein were judged to beantibody-positive.

Spleens were excised from those mice which were thus judged to bepositive with respect to antibody against the purified mucusglycoprotein antigen and an aqueous suspension of single splenic cellswas prepared therefrom for use in cell fusion.

(3) Preparation of mouse myeloma cells

8-azaguanine-resistant mouse myeloma cells Sp2/O-Ag14 were cultured at37° C. in normal medium [RPMI 1640 (Nissui Pharmaceutical Co., Ltd. )(10.2 g/l) medium supplemented with sodium hydrogen carbonate (2.2 g/l),L-glutamine (0.3 g/l), gentamycin (40 mg/l) and fetal calf serum (10%V/V)], using a carbonic acid gas incubator.

(4) Cell fusion and cultivation of hybridomas

The splenic cells and mouse myeloma cells Sp2/O-Ag14 obtained above weremixed together at a cell count ratio of 10:1 and then subjected to cellfusion while adding a fusing solution [polyethylene glycol 4,000 (0.5 g)in dimethyl sulfoxide (0.05 ml) and PBS (0.5 ml)] to the mixture withgentle stirring. After incubation at 37° C. for 90 seconds, a medium[RPMI 1640 (Nissui Pharmaceutical Co., Ltd. ) (10.2 g/l) mediumsupplemented with sodium hydrogen carbonate (2.2 g/l), L-glutamine (0.3g/l) and gentamycin (40 mg/l)] was slowly added thereto to make up thetotal fluid volume to 40 ml. After centrifugation at 1,100 rpm for 10minutes, the supernatant was removed and HAT (hypoxanthine aminopoterinthymidine) medium [RPMI 1640 (Nissui Pharmaceutical Co., Ltd. ) (10.2g/l) medium supplemented with sodium hydrogen carbonate (2.2 g/l),L-glutamine (0.3 g/l), gentamycin (40 mg/l), fetal calf serum (20% V/V),hypoxanthine (100 μmol/l), aminopterin (0.4 μmol/l) and thymidine (16μmol/l)] to suspend the cells therein in a gentle manner. The suspensionwas added to each well of a 96-well culture plate and the suspension wascultivated in an incubator containing 5% carbonic acid gas.

(5) Screening and cloning

From the wells of the above-described 96-well culture plate where thegrowth of fused cells in the form of colonies were observed, there weretaken aliquots of each culture supernatant. Screening for hybridomasproducing antibody capable of reacting with the purified mucusglycoprotein antigen was performed by the ELISA as described below.There were thus selected HIK-22 and HIK-108. Further screening wascarried out with respect to the antibodies produced by HIK-22 andHIK-108, using the immunohistochemical staining method as mentionedbelow. There was thus selected HIK-108 as hybridoma producing anantibody with reactivity with gastric gland mucous cells as well asmucus secreted thereby. The HIK-108 was subcultured for one week in HT(hypoxanthine thymidine) medium [RPMI 1640 (Nissui Pharmaceutical Co.,Ltd.) (10.2 g/l) medium supplemented with sodium hydrogen carbonate.(2.2 g/l), L-glutamine (0.3 g/l), gentamycin (40 mg/l), fetal calf serum(15% V/V), hypoxanthine (100 μmol/l) and thymidine (16 μmol/l)] and foranother week in normal medium [RPMI 1640 (Nissui Pharmaceutical Co.,Ltd.) (10.2 g/l) medium supplemented with sodium hydrogen carbonate (2.2g/l), L-glutamine (0.3 g/l), gentamycin (40 mg/l) and fetal calf serum(10% V/V)], and two limiting dilution subclonings were conducted to givethree hybridomas, HIK-1081, HIK-1082 and HIK-1083. These hybridomas wereindividually subcultured for 4 weeks in the normal medium and eachhybridoma culture supernatant was analyzed by the ELISA method asmentioned below. There was thus selected HIK-1083 (deposited in theNational Institute of Bioscience and Human-Technology (NIBH), Agency ofIndustrial Science and Technology with the accession number P-13622) asthe hybridoma giving the culture supernatant showing the most intensecolor development.

ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05Msodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at aconcentration of 1 μg/ml. 100 μl of the solution was added to each wellof a microplate adapted for use in ELISA (Corning) and was allowed tostand overnight at 4° C. Each well was washed three times with 0.05%TWEEN 20 in PBS (PBS-TWEEN), filled with 2% skim milk in PBS and thenallowed to stand for 1 hour. The wells were washed three times withPBS-TWEEN and 100 μl/well of hybridoma culture supernatant obtained in(5) above was added to each well. After incubation for 1 hour, the wellswere washed three times with PBS-TWEEN, and 100 μl/well of a 10,000-folddilution in PBS of peroxidase-conjugated goat anti-mouse immunoglobulinsantibody (Tago, Inc.) was added as secondary antibody to each well andallowed to stand for 1 hour. The wells were washed three times withPBS-TWEEN and 100 μl of an ABTS-H₂ O₂ peroxidase substrate solution(Kirkegaard & Perry Laboratories) was added to each well. After reactionat room temperature for 30 minutes, the optical density at 415 nm wasmeasured for each well using a microplate reader. Those wells wereselected which showed a more intense color development than the wellstreated in the same manner as described above except that the myelomacell culture supernatant obtained in (3) above was added instead of thehybridoma culture supernatant. The samples corresponding to the soselected color-developing wells were judged to be positive.

Immunohistochemical staining: Human gastric mucosa was fixed withformalin, embedded in paraffin and sliced into 4 μm-thick sections usinga microtome. Each of the thus prepared sections was fixed on a slideglass. Each slide glass was dipped in xylene for deparaffinization andthen in methanol containing 0.3% hydrogen peroxide for 30 minutes andthen washed by dipping in PBS for 30 minutes. Subsequently, 10% rabbitnormal serum (Nichirei Corporation) was applied to the thus washedsection on each slide glass. After incubation for 1 hour, the sectionswere washed in PBS. Samples of hybridoma culture supernatant wereapplied onto the washed sections. After incubation for 1 hour, thesections were washed in PBS. Biotin-labeled rabbit anti-mouseIgG+IgA+IgM (H+L) (Nichirei Corporation) was applied onto each washedsection. After incubation for 1 hour, the sections were washed in PBS.Peroxidase-conjugated streptavidin (Nichirei Corporation) was thenapplied onto each washed section. After incubation for 1 hour, thesections were washed in PBS. Subsequently, each washed section wasdipped for about 4 minutes in 0.05M Tris-HCl buffer, pH 7.6, containing0.02% diaminobenzidine (Dojindo Laboratories) and 0.005% aqueoushydrogen peroxide, for color development.

(6) Identification of isotype

The monoclonal antibody according to the present invention was analyzedfor globulin class by ELISA using an isotyping kit (PharMingen). Thus,monoclonal rat anti-mouse IgG₁, monoclonal rat anti-mouse IgG_(2a),monoclonal rat anti-mouse IgG_(2b), monoclonal rat anti-mouse IgG₃,monoclonal rat anti-mouse IgM, monoclonal rat anti-mouse IgA, monoclonalrat anti-mouse IgL (κ) and monoclonal rat anti-mouse IgL (λ) were usedand each reagent was diluted 1:5 with coating buffer. Each 50 μl/well ofthe dilutions was poured into the wells of a microplate adapted for usein ELISA (Corning) and the wells were allowed to stand overnight at 4°C. Each well was washed three times with 0.05% TWEEN 20 in PBS(PBS-TWEEN), filled with 2% skim milk in PBS and then allowed to standfor 1 hour. The wells were washed three times with PBS-TWEEN and culturesupernatant from a 3-day culture of HIK-1083 (Accession number P-13622)in a normal medium [RPMI 1640 (Nissui Pharmaceutical Co., Ltd. ) (10.2g/l) medium supplemented with sodium hydrogen carbonate (2.2 g/l),L-glutamine (0.3 g/l), gentamycin (40 mg/l) and fetal calf serum (10%V/V)] was dispensed in 50 μl portions into the wells. After 1-hourincubation, the wells were washed three times with PBS-TWEEN and 50 μlof an alkaline phosphatase-labeled polyclonal rat anti-mouse Igs reagentwas added to each well. The wells were allowed to stand for 1 hour andthen washed three times with PBS-TWEEN. To each well was added 50 μl ofa substrate solution prepared by dissolving one pNPP(p-Nitrophenylphosphate) tablet in 5 ml of a solvent for the substrate.

After incubation at room temperature for 30 minutes, the microplate wasvisually observed to find that the only two wells pretreated with themonoclonal rat anti-mouse IgM reagent and the monoclonal rat anti-mouseIgL (κ) reagent, respectively, showed an intense-yellow coloration, theother wells being colorless and transparent. On the basis of theseresults, the monoclonal antibody of the present invention was identifiedas an IgM antibody with the light chain being a κ chain.

(7) Preparation of monoclonal antibody

Six-week-old female BALB/c mice weighing 19-21 g were intraperitoneallyadministered with 0.5 ml/animal of 2,6,10,14-tetramethylpentadecane(pristane) and then raised for 10-14 days. These animal were thenintraperitoneally administered with 5×10⁶ cells/animal of theabove-mentioned hybridoma strain HIK-1083 (Accession number P-13622).After 10-21 days, the accumulated ascites fluid was collected andsubjected to centrifugation (12,000 rpm/20 minutes/4° C.).

The supernatant was then separated and ammonium sulfate was added to 40%saturation for salting out. The mixture was subjected to centrifugation(12,000 rpm/20 minutes/4° C.). The resulting sediment was dissolved inPBS and then dialyzed against PBS at 4° C. for 2 days and the dialyzatewas loaded onto an affinity column as follows. Thus pig gastric mucusglycoprotein (Sigma) was dissolved in 0.1M phosphate buffer, pH 7.8,containing 0.5M NaCl and the solution was mixed with CNBr-ActivatedSEPHAROSE 4B, agarose gel beads (Pharmacia) swelled with the samebuffer. A column was packed with the mixture and then washed with thesame buffer. The components of the above-mentioned dialyzate wereadsorbed onto the column and the so adsorbed dialyzate components wereeluted with 0.2M glycine-HCl buffer, pH 2.0, containing 0.5M NaCl. 3MTris-HCl buffer, pH 8.5, was added to the eluate and the resultingmixture was used for the following labeling reaction with horseradishperoxidase.

(8) Labeling of monoclonal antibody

Horseradish peroxidase (Amano Pharmaceutical Co., Ltd.) was dissolved indistilled water and a 0.1M aqueous solution of sodium metaperiodate wasadded and the mixture was incubated for 10 minutes. Ethylene glycol wasadded to the reaction solution and the mixture was desalted using aSEPHADEX G-25, dextran gel beads bridged with epichlorohydrin column(Pharmacia) to prepare a periodate-processed peroxidase solution. Theperiodate-processed peroxidase solution was mixed with theabove-mentioned monoclonal antibody purified from the mouse ascitesfluid by using the affinity column. The mixture was incubated at roomtemperature for 2 hours and after a 0.1M aqueous solution of sodiumborohydride was added the mixture was incubated for a further 2 hours.The resulting reaction solution was fractionated into 35 fractions byusing a SEPHACRYL S-200 HR, allyl dextran gel beads bridged withN,N'-methylene bis-acrylamide (Pharmacia) and each fraction was analyzedby the ELISA method as described below. Four fractions which gave colordevelopment were combined and frozen before storage.

ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05Msodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at aconcentration of 1 μg/ml. 100 μl of the solution was added to each wellof a microplate adapted for use in ELISA (Corning) and was allowed tostand overnight at 4° C. Each well was washed three times with 0.05%TWEEN 20 in PBS (PBS-TWEEN), filled with 2% skim milk in PBS and thenallowed to stand for 1 hour. The wells were washed three times withPBS-TWEEN and the column eluate sample (each fraction) was dispensed in100 μl aliquots into the wells, followed by incubation for 1 hour. Thewells were washed three times with PBS-TWEEN and 100 μl of an ABTS-H₂ O₂peroxidase substrate solution (Kirkegaard & Perry Laboratories) wasadded to each well. After incubation at room temperature for 30 minutes,the optical density at 415 nm was measured for each well using amicroplate reader.

Example 2 Antigenic specificity of monoclonal antibody

(1) Confirmation of antigenic specificity by immunohistochemicalstaining

The reactivity of the monoclonal antibody obtained in Example 1 withhuman gastric mucosa was studied by immunohistochemical staining. Thushuman gastric and duodenal mucosae were fixed with formalin, embedded inparaffin and sliced into 4 μm-thick sections using a microtome. Each ofthe thus prepared sections was fixed on a slide glass. Each slide glasswas dipped in xylene for deparaffinization and then in methanolcontaining 0.3% hydrogen peroxide for 30 minutes and then washed bydipping in PBS for 30 minutes. Subsequently, 10% rabbit normal serum(Nichirei Corporation) was applied onto the thus washed section on eachslide glass. After incubation for 1 hour, the sections were washed inPBS. Culture supernatant from a 3-day culture of the hybridoma HIK-1083(Accession number P-13622) in normal medium [RPMI 1640 (NissuiPharmaceutical Co., Ltd.) (10.2 g/l) medium supplemented with sodiumhydrogen carbonate (2.2 g/l), L-glutamine (0.3 g/l), gentamycin (40mg/l) and fetal calf serum (10% V/V)] was then applied onto the washedsection on each slide glass. After incubation for 1 hour, the sectionswere washed in PBS. Biotin-labeled rabbit anti-mouse IgG+IgA+IgM (H+L)(Nichirei Corporation) was applied onto each washed section. Afterincubation for 1 hour, the sections were washed in PBS.Peroxidase-conjugated streptavidin (Nichirei Corporation) was thenapplied onto each washed section. After incubation for 1 hour, thesections were washed in PBS. Subsequently, each washed section wasdipped for about 4 minutes in 0.05M Tris-HCl buffer, pH 7.6, containing0.02% diaminobenzidine (Dojindo Laboratories) and 0.005% aqueoushydrogen peroxide, for color development. The results were as shown inTable 1.

These results show that human gastric gland-type mucus (class III mucus)can be specifically detected by using the monoclonal antibody obtainedin Example 1.

                  TABLE 1                                                         ______________________________________                                        Gastric mucosa                                                                           Corpus      Mucus gel layer                                                                             +                                                               Surface mucous cell                                                                         -                                                               Mucous neck cell                                                                            +                                                   Antrum      Mucus gel layer                                                                             +                                                               Surface mucous cell                                                                         -                                                               Pyloric gland cell                                                                          +                                        Duodenal mucosa        Villus epithelium                                                                           -                                                               Brunner's gland                                                                             +                                        ______________________________________                                         N.B.                                                                          +: positive to staining;                                                      -: negative to staining                                                  

(2) Analysis of components

The mucus glycoprotein antigen with which the monoclonal antibodyobtained in Example 1 reacted specifically was analyzed.

(a) Preparation of mucus glycoprotein decomposition product

Pig gastric mucus glycoprotein (Sigma) was subjected, in accordance withthe procedure as described in Carlson, Don M. (1968) J. Biol. Chem. 243,616-626, to 24-hour heat treatment at 50° C. in 0.05M aqueous sodiumhydroxide solution containing 1M sodium borohydride.

(b) Separation and purification of the decomposition product mentionedabove

The reaction solution obtained in (a) above was cooled down to roomtemperature and applied to a column TOYOPEARL HW-50S, a hydrophilicvinyl polymer based filler (Tosoh Corporation) pre-equilibrated with0.1N acetic acid. The column was eluted with 0.5N acetic acid and theeluate was fractionated into 40 fractions. The resultant fractions wereassayed for antigenic activity by the Competitive ELISA as describedbelow and the fractions with antigenic activity which were eluted latestwere selected. The thus selected fractions were further fractionatedinto 70 fractions by means of HPLC using a TSK gel NH₂ -60 a silica gelfiller chemically bonded with amino propyl groups column (TosohCorporation) and each fraction was assayed for antigenic activity by theCompetitive ELISA as described below. Among the resultant 70 fractions,three fractions showing stronger antigenic activity were selected andthese three fractions were combined and evaporated to dryness in vacuo.

Competitive ELISA: The purified mucus glycoprotein antigen was dissolvedin 0.05M sodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at aconcentration of 2 μg/ml. 100 μl of the solution was added to each wellof a microplate adapted for use in ELISA (Corning) and was allowed tostand overnight at 4° C. Each well was washed three times with 0.05%TWEEN 20 in PBS (PBS-TWEEN), filled with 2% skim milk in PBS and thenallowed to stand for 1 hour. The wells were washed three times withPBS-TWEEN. In a separate container, 50 μl of the sample, i.e. theabove-mentioned column eluate of mucus glycoprotein antigendecomposition product, 25 μl of culture supernatant from a 3-day cultureof hybridoma HIK-1083 (Accession number P-13622) in normal medium [RPMI1640 (Nissui Pharmaceutical Co., Ltd. ) (10.2 g/l) medium supplementedwith sodium hydrogen carbonate (2.2 g/l), L-glutamine (0.3 g/l),gentamycin (40 mg/l) and fetal calf serum (10% V/V)] and 25 μl of a4-fold concentrate of PBS were mixed together and the mixture wasallowed to stand for 2 hours. These mixture were added to the washedwells. After incubation for 1 hour, the wells were washed three timeswith PBS-TWEEN, and 100 μl of a 1:10,000 dilution in PBS ofperoxidase-conjugated goat anti-mouse immunoglobulins antibody (Tago,Inc.) was added as secondary antibody to each well. After 1 hour ofincubation, the wells were washed three times with PBS-TWEEN and 100 μlof an ABTS-H₂ O₂ peroxidase substrate solution (Kirkegaard & PerryLaboratories) was added to each well. After 30 minutes of incubation atroom temperature, the optical density at 415 nm was measured for eachwell using a microplate reader. Those samples corresponding to the wellswhich gave lower optical density than that of the reaction solution inthe well where the above-described procedure was performed usingdistilled water instead of the samples were judged as ones havingantigenic activity.

(c) Analysis of product obtained by evaporation to dryness in vacuo

The product obtained in (b) above by evaporation to dryness in vacuo wastrimethylsilylated in accordance with the method of Sweeley et al.(Sweeley, C. C. (1963) J. Am. Chem. Soc. 85, 2497-2507) and thenanalyzed by gas chromatography. Thus, the above-mentioned product wassubjected to heat treatment in methanol containing 3% HCl and silvercarbonate was added to the reaction solution to adjust the pH to 5.Subsequently acetic anhydride was added to the reaction solution and themixture was allowed to stand overnight at room temperature. The mixturewas then centrifuged at 2,000 rpm for 5 minutes and the supernatant wasremoved and evaporated to dryness in vacuo. A TRI-SIL,hexamethyldisilazane, trimethylchlorosilane and high purity gradepyridine (2:1:10) reagent (Pierce) was added to dissolve in theresultant concentrated residue. After incubation, aliquots of thereaction solution were injected into a Model "GC7A gas chromatograph(Shimazu Corporation) equipped with an OV-1 (dimethyl silicone gum)capillary column (GL Science Inc.) measuring 2.5 m in length and 0.25 mmin inner diameter, followed by detection using an FID (frame-ionizationdetector). The detected peaks were identified by comparison withchromatograms obtained with standard substances under the sameanalytical conditions. The standard substances were prepared by reactinga TRI-SIL reagent (Pierce) with fucose, galactose, glucose, mannose,N-acetylgalactosamine or N-acetylglucosamine. Furthermore,N-acetylgalactosaminitol standard substance was prepared by reduction ofN-acetylgalactosamine in 0.2M borate buffer, pH 9, containing sodiumborohydride, followed by reaction with a TRI-SIL reagent (Pierce). Therewere thus detected, from the concentrated residue of the above describedfractions with antigenic activity, N-acetylgalactosaminitol,N-acetylglucosamine, fucose and galactose.

Example 3 Reactivity with sugars

For each of methyl 2-acetamido-2-deoxy-α-D-glucopyranoside (Sigma) andmethyl 2-acetamido-2-deoxy-β-D-glucopyranoside (Sigma), a 1 mg/mlsolution in distilled water was prepared and two-fold serial dilutionswere prepared therefrom using distilled water. The reactivity of themonoclonal antibody obtained in Example 1 with these aqueous solutionswere examined by the Competitive ELISA as described below. As a result,the methyl 2-acetamido-2-deoxy-α-D-glucopyranoside decreased the opticaldensity in dependence upon its concentration and hence the reactivitytherewith was observed, whereas no such concentration-dependent changein the optical density was found with the methyl2-acetamido-2-deoxy-β-D-glucopyranoside.

Competitive ELISA: The purified mucus glycoprotein antigen was dissolvedin 0.05M sodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at aconcentration of 2 μg/ml. 100 μl of the solution was added to each wellof a microplate adapted for use in ELISA (Corning) and was allowed tostand overnight at 4° C. Each well was washed three times with 0.05%TWEEN 20 in PBS (PBS-TWEEN), filled with 2% skim milk in PBS and thenallowed to stand for 1 hour. The wells were washed three times withPBS-TWEEN. In a separate container, 50 μl of the sample, i.e. the abovedescribed aqueous solution, 25 μl of culture supernatant from a 3-dayculture of hybridoma HIK-1083 (Accession number P-13622) in normalmedium [RPMI 1640 (Nissui Pharmaceutical Co., Ltd.) (10.2 g/l) mediumsupplemented with sodium hydrogen carbonate (2.2 g/l), L-glutamine (0.3g/l), gentamycin (40 mg/l) and fetal calf serum (10% V/V)] and 25 μl of4-fold concentrate of PBS were mixed together and the mixture wasallowed to stand for 2 hours. These mixtures were added to the washedwells. After incubation for 1 hour, the wells were washed three timeswith PBS-TWEEN, and 100 μl of a 1:10,000 dilution in PBS ofperoxidase-conjugated goat anti-mouse immunoglobulins antibody (Tago,Inc.) was added as secondary antibody to each well. After 1 hour ofincubation, the wells were washed three times with PBS TWEEN and 100 μlof an ABTS-H₂ O₂ peroxidase substrate solution (Kirkegaard & PerryLaboratories) was added to each well. After 30 minutes of incubation atroom temperature, the optical density at 415 nm was measured for eachwell using a microplate reader.

We claim:
 1. An IgM class monoclonal antibody which specifically reactswith Class III mucus glycoproteins and which does not react with humangastric surface mucous cells or the mucus glycoproteins secretedthereby.
 2. The monoclonal antibody of claim 1, wherein the IgM classmonoclonal antibody is produced by a hybridoma deposited in the NationalInstitute of Bioscience and Human-Technology (NIBH), Agency ofIndustrial Science and Technology with the accession number P-13622. 3.A hybridoma which produces an IgM class monoclonal antibody withspecific reactivity with Class III mucus glycoproteins and no reactivitywith human gastric surface mucous cells or the mucus secreted thereby.4. A hybridoma deposited in the National Institute of Bioscience andHuman-Technology (NIBH), Agency of Industrial Science and Technologywith the accession number P-13622.
 5. A method of determining thepresence or amount of Class III mucus glycoproteins comprising:obtaininga sample of human body fluid; contacting said sample with the monoclonalantibody of claim 1; measuring formation of any specific bindingcomplexes comprising the monoclonal antibody of claim 1; and correlatingthe presence or amount of said specific binding complexes to thepresence or amount of said class III mucus glycoproteins in said sample.